Aminoglycoside antibiotics like gentamicin are essential for battling life-threatening bacterial infections. Aminoglycosides also cause permanent deafness/balance disorders and nephrotoxicity in more than 120,000 individuals each year in the US, particularly in infants and premature babies. The long-term goal of this research is to prevent aminoglycoside-induced ototoxicity by modulating the functions of gentamicin-binding proteins crucial for ototoxicity, to preserve auditory function. In this proposal, we seek to identify gentamicin-binding proteins, and determine their distribution in the inner ear. We hypothesize that there are gentamicin-binding proteins in the inner ear, and that these proteins retain gentamicin in specific types of cells, contributing to cytotoxicity in vitro and pathogenesis in vivo. The specific aims of this project are: First, to identify gentamicin-binding proteins specific in kidney proximal tubule and cochlear cells. We will use a gentamicin-agarose pull-down assay and SDS-polyacrylamide gel electrophoresis to isolate such proteins from kidney proximal tubule and cochlear cell lines, and determine their identity using mass spectrometry (Aim 1). Second, to determine the distribution of gentamicin-binding proteins, including HSP73 and calreticulin, in the inner ear by immunocytochemistry and confocal microscopy. We will also determine if these proteins are co-localized with systemically-administered gentamicin, using fluorescently-tagged gentamicin or untagged gentamicin localized by gentamicin immunocytochemistry (Aim 2). If we can identify gentamicin-binding proteins that sequester the drug within cells that then induce cytotoxicity, we can develop new strategies to inhibit their interactions, and attenuate drug retention. Alternatively, if gentamicin-binding inhibits the functions of these proteins, we could use pharmacological activators of these proteins to overcome gentamicin-induced inhibition. Future studies will determine if gentamicin inhibits their interactions with physiological binding molecules/proteins, and if expression levels of gentamicin-binding proteins change during sustained treatment with gentamicin. Identification of gentamicin-binding proteins and their intracellular mechanisms that gentamicin interferes with to initiate cell death will provide new insight for clinicians to screen patients for pre-existing conditions and medications that elevate the risk of aminoglycoside toxicity and deafness. Identification of gentamicin-binding proteins that play a major role in gentamicin- induced ototoxicity is crucial in understanding how gentamicin induces ototoxicity and deafness. The proposed research will enable us to develop strategies to prevent interactions between gentamicin and gentamicin-binding proteins, and to overcome gentamicin-induced inhibition by pharmacologically activating the proteins. These strategies will lead to ototoxicity and deafness prevention.